Mechanical Perturbation of Bacterial Translation Fidelity PROJECT SUMMARY The objective of this proposal is to interrogate a model of protein synthesis that accounts for collisions between ribosomes as they translate messages. Nearly all of our mechanistic understanding of translation has been presented in the context of single ribosomes staged in isolated scenarios. What is missing from this picture is the reality that most ribosomes translate messages as members of polysomes. Depending on the rates of initiation, translocation, and termination, ribosomes are likely to come into contact with one another as they traverse mRNAs. In the event a ribosome becomes stalled, either because it has been poisoned by an antibiotic, or because it encountered a difficult decoding region, uncompromised ribosomes following it on the same mRNA will push forward against it. We believe this mechanical perturbation contributes to errors in translation and that there are conserved mechanisms to alleviate this interference. This proposal presents three aims that address different aspects of this model. In the first aim, the decoding fidelity at established mistranslation motifs will be determined as a function of ribosome crowding. The prediction is that the thrust of trailing ribosomes will increase translation errors because the imparted energy disrupts the stalled ribosomes. Also, we will test to see if a conserved bacterial fidelity factor, ribosomal protein L9, has any role in alleviating this stress. In the second aim, we address whether ribosomes assembled in translationally-compromised cells are themselves partially defective at decoding because they were not matured completely. Again, we will test to see if L9 has an impact on their translation performance. Finally, the third aim will directly evaluate the idea that L9 dampens the forward progression of trailing ribosomes against stalled ones. These will be the the first mechanistic tests of L9 function and they will address a long-standing question of how a factor located far from the decoding center can have such a strong influence on fidelity. Collectively, these experiments will reframe our mechanistic understanding of translation in many systems, especially those that involve transient or prolonged stalling. Our goal is to mechanistically understand a complete picture of bacterial translation and the challenges ribosomes face as they overcome biochemical hurdles. Our studies will open a new door for therapeutic strategies aimed at specifically disrupting bacterial translation fidelity, which could revive a shelved class of potent antibiotics that promote translation errors.